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3D cell culture has emerged as a promising approach to replicate the complex behaviors of cells within living organisms. This study aims to analyze spatiotemporal behavior of the morphological characteristics of cell structure at multiscale in 3D scaffold-free spheroids using chondrogenic progenitor ATDC5 cells. Over a 14-day culture period, it exhibited cell hypertrophy in the spheroids regarding cellular and nuclear size as well as changes in morphology. Moreover, biological analysis indicated a signification up-regulation of normal chondrocyte as well as hypertrophic chondrocyte markers, suggesting early hypertrophic chondrocyte differentiation. Cell nuclei underwent changes in volume, sphericity, and distribution in spheroid over time, indicating alterations in chromatin organization. The ratio of chromatin condensation volume to cell nuclear volume decreased as the cell nuclei enlarged, potentially signifying changes in chromatin state during hypertrophic chondrocyte differentiation. Our image analysis techniques in this present study enabled detailed morphological measurement of cell structure at multi-scale, which can be applied to various 3D culture models for in-depth investigation. [ABSTRACT FROM AUTHOR]

Abstract Background The relationship between thyroid hormone (TH) levels in vivo and osteoarthritis (OA) remains inconclusive. This study aims to investigate the association between TH levels and OA, analyze the effect of triiodothyronine on hypertrophic chondrocyte differentiation and OA progression, and identify potential target genes of triiodothyronine in OA to evaluate its diagnostic value. Methods Two-sample mendelian randomization method was used to probe the causal links between hyperthyroidism and OA. Differentially expressed genes (DEGs) from two RNA-sequencing data in Gene Expression Omnibus (GSE199847 and GSE114007) and enrichment analysis of DEGs (166 commonly upregulated genes and 71 commonly downregulated genes of GSE199847 and GSE114007) was performed to analyze the effect of triiodothyronine (T3) on hypertrophic chondrocyte differentiation and OA. C28/I2 cells treated with T3 and reverse transcription and quantitative real-time polymerase chain reaction were used to validate T3 targeted genes. The diagnostic performance of target genes was assessed by the receiver operating characteristic (ROC) curve and area under the curve (AUC). Results There was a positive causal association between hyperthyroidism and OA (IVW result, OR = 1.330, 95% CI 1.136–1.557, P = 0.0004). Weighted median and Weighted mode analysis also demonstrated that hyperthyroidism had a positive causal association with OA (p 1). Bioinformatics analysis indicated T3 can partially induce the emergence of late hypertrophic chondrocyte and promote OA through extracellular matrix organization, blood vessel development, skeletal system development and ossification. Post-T3 treatment, MAFB, C1QTNF1, COL3A1 and ANGPTL2 were significantly elevated in C28/I2 cells. ROC curves in GSE114007 showed that AUC of all above genes were ≥ 0.7. Conclusions This study identified that hyperthyroidism has a positive causal association with OA by MR analysis. T3 induced hypertrophic chondrocytes promote OA progression by upregulating genes such as MAFB, C1QTNF1, COL3A1 and ANGPTL2, which can also serve as OA diagnosis.

Osteoarthritis (OA) is a chronic degenerative disease that affects the whole joint, especially the knee joint. Its main features include articular cartilage defects and osteophyte formation, and it is common in middle-aged and elderly people. Although the pathogenesis of OA is not fully understood, mechanical factors, inflammation and immune abnormalities can affect joint tissue metabolism and destroy cartilage and bone homeostasis. Cartilage calcification is closely related to chondrocyte hypertrophy, differentiation and bone sclerosis in OA, which is manifested as pathological calcification of cartilage matrix. Chondrocytes in OA may change from a state of maintaining cartilage matrix balance to a state of promoting cartilage destruction and calcification. Inflammatory factors such as TNF-α and IL-1β promote this phenotypic shift, accelerating matrix degradation and calcium salt deposition. The change of calcium signal and an important factor of angiogenesis and promote cartilage calcification. Chondrocyte hypertrophy plays a crucial role in the pathogenesis and progression of OA, characterized by complex interactions with cartilage calcification, subchondral bone sclerosis, as well as chondrocyte proliferation, apoptosis, matrix remodeling, and signaling cascades. The degree of chondrocyte hypertrophy exhibits a positive correlation with the severity of OA. Furthermore, structural changes in the articular cartilage are associated with factors including reduced cartilage collagen synthesis or the activation by degradative enzymes. Regulatory mechanisms governing chondrocyte hypertrophy and cartilage calcification, alongside the identification of pertinent genes, represent pivotal areas for future investigation. This research will further elucidate the pathogenesis of OA and lay the groundwork for devising therapeutic strategies.

Background: The relationship between thyroid hormone (TH) levels in vivo and osteoarthritis (OA) remains inconclusive. This study aims to investigate the association between TH levels and OA, analyze the effect of triiodothyronine on hypertrophic chondrocyte differentiation and OA progression, and identify potential target genes of triiodothyronine in OA to evaluate its diagnostic value. Methods: Two-sample mendelian randomization method was used to probe the causal links between hyperthyroidism and OA. Differentially expressed genes (DEGs) from two RNA-sequencing data in Gene Expression Omnibus (GSE199847 and GSE114007) and enrichment analysis of DEGs (166 commonly upregulated genes and 71 commonly downregulated genes of GSE199847 and GSE114007) was performed to analyze the effect of triiodothyronine (T3) on hypertrophic chondrocyte differentiation and OA. C28/I2 cells treated with T3 and reverse transcription and quantitative real-time polymerase chain reaction were used to validate T3 targeted genes. The diagnostic performance of target genes was assessed by the receiver operating characteristic (ROC) curve and area under the curve (AUC). Results: There was a positive causal association between hyperthyroidism and OA (IVW result, OR = 1.330, 95% CI 1.136–1.557, P = 0.0004). Weighted median and Weighted mode analysis also demonstrated that hyperthyroidism had a positive causal association with OA (p < 0.05, OR > 1). Bioinformatics analysis indicated T3 can partially induce the emergence of late hypertrophic chondrocyte and promote OA through extracellular matrix organization, blood vessel development, skeletal system development and ossification. Post-T3 treatment, MAFB, C1QTNF1, COL3A1 and ANGPTL2 were significantly elevated in C28/I2 cells. ROC curves in GSE114007 showed that AUC of all above genes were ≥ 0.7. Conclusions: This study identified that hyperthyroidism has a positive causal association with OA by MR analysis. T3 induced hypertrophic chondrocytes promote OA progression by upregulating genes such as MAFB, C1QTNF1, COL3A1 and ANGPTL2, which can also serve as OA diagnosis. [ABSTRACT FROM AUTHOR]

Endochondral ossification is the process of bone formation derived from growing cartilage duringskeletal development. In previous studies, we provoked the osteocyte differentiation of osteoblast precursor cells under a three‐dimensional (3D) culture model. To recapitulate the endochondral ossification, the present study utilized the self‐organized scaffold‐free spheroid model reconstructed by pre‐chondrocyte cells. Within 2‐day cultivation in the absence of the chemically induced chondrogenesis supplements, the chondrocyte marker was greatly expressed in the inner region of the spheroid, whereas the hypertrophic chondrocyte marker was strongly detected in the surface region of the spheroid. Notably, we found out that the gene expression levels of osteocyte markers were also greatly upregulated compared to the conventional 2D monolayer. Moreover, after long‐term cultivation for 28 days, it induced morphological changes in the spheroid, such as cellular hypertrophy and death. In this study, in order to recapitulate the initial stage of the endochondral ossification, we highlighted the potentials of the 3D culture method to drive the hypertrophic chondrocyte differentiation of the pre‐chondrocyte cells. [ABSTRACT FROM AUTHOR]

Endochondral bone formation is an important pathway in fracture healing, involving the formation of a cartilaginous soft callus and the process of cartilage-to-bone transition. Failure or delay in the cartilage-to-bone transition causes an impaired bony union such as nonunion or delayed union. During the healing process, multiple types of cells including chondrocytes, osteoprogenitors, osteoblasts, and endothelial cells coexist in the callus, and inevitably crosstalk with each other. Hypertrophic chondrocytes located between soft cartilaginous callus and bony hard callus mediate the crosstalk regulating cell-matrix degradation, vascularization, osteoclast recruitment, and osteoblast differentiation in autocrine and paracrine manners. Furthermore, hypertrophic chondrocytes can become osteoprogenitors and osteoblasts, and directly contribute to woven bone formation. In this review, we focus on the roles of hypertrophic chondrocytes in fracture healing and dissect the intermingled crosstalk in fracture callus during the cartilage-to-bone transition.

Background: Progressive maturation of growth plate chondrocytes drives long bone growth during endochondral ossification. Signals from the epidermal growth factor receptor (EGFR), and from bone morphogenetic protein‐2 (BMP2), are required for normal chondrocyte maturation. Here, we investigated cross‐talk between EGFR and BMP2 signals in developing and adult growth plates. Results: Using in vivo mouse models of conditional cartilage‐targeted EGFR or BMP2 loss, we show that canonical BMP signal activation is increased in the hypertrophic chondrocytes of EGFR‐deficient growth plates; whereas EGFR signal activation is increased in the reserve, prehypertrophic and hypertrophic chondrocytes of BMP2‐deficient growth plates. EGFR‐deficient chondrocytes displayed increased BMP signal activation in vitro, accompanied by increased expression of IHH, COL10A1, and RUNX2. Hypertrophic differentiation and BMP signal activation were suppressed in normal chondrocyte cultures treated with the EGFR ligand betacellulin, effects that were partially blocked by simultaneous treatment with BMP2 or a chemical EGFR antagonist. Conclusions: Cross‐talk between EGFR and BMP2 signals occurs during chondrocyte maturation. In the reserve and prehypertrophic zones, BMP2 signals unilaterally suppress EGFR activity; in the hypertrophic zone, EGFR and BMP2 signals repress each other. This cross‐talk may play a role in regulating chondrocyte maturation in developing and adult growth plates. Key Findings: epidermal growth factor receptor, EGFR, bone morphogenetic protein, BMP2, BMP, chondrocyte maturation, hypertrophic chondrocyte, growth plate, betacellulin, BTC [ABSTRACT FROM AUTHOR]

Abstract Rheumatoid arthritis (RA) is characterized by chronic joint inflammation, which forms pannus with bone destruction. Bony ankylosis is also observed following inflammation; however, the mechanism behind this aberrant bone formation in RA had remained unclear. Based on our recent findings obtained using a novel arthritis model called D1BC mouse, we found that synovial fibroblasts in pannus consist of at least three different populations with the osteochondrogenic lineage being predominant. We also found endochondral ossification like that in embryonic bone development adjacent to invasive synovial fibroblasts. Such ectopic endochondral ossification leads to the failure of bone repair and results in ankylosis. In this review, we describe the character of synovial fibroblasts toward the osteochondrogenic lineage and ectopic endochondral ossification in an inflammatory arthritis mouse model.

Cellular plasticity refers to the ability of cell fates to be reprogrammed given the proper signals, allowing for dedifferentiation or transdifferentiation into different cell fates. In vitro, this can be induced through direct activation of gene expression, however this process does not naturally occur in vivo. Instead, the microenvironment consisting of the extracellular matrix (ECM) and signaling factors, directs the signals presented to cells. Often the ECM is involved in regulating both biochemical and mechanical signals. In stem cell populations, this niche is necessary for maintenance and proper function of the stem cell pool. However, recent studies have demonstrated that differentiated or lineage restricted cells can exit their current state and transform into another state under different situations during development and regeneration. This may be achieved through (1) cells responding to a changing niche; (2) cells migrating and encountering a new niche; and (3) formation of a transitional niche followed by restoration of the homeostatic niche to sequentially guide cells along the regenerative process. This review focuses on examples in musculoskeletal biology, with the concept of ECM regulating cells and stem cells in development and regeneration, extending beyond the conventional concept of small population of progenitor cells, but under the right circumstances even “lineage-restricted” or differentiated cells can be reprogrammed to enter into a different fate.

According to the general understanding, the chondrocyte lineage terminates with the elimination of late hypertrophic cells by apoptosis in the growth plate. However, recent cell tracking studies have shown that murine hypertrophic chondrocytes can survive beyond “terminal” differentiation and give rise to a progeny of osteoblasts participating in endochondral bone formation. The question how chondrocytes convert into osteoblasts, however, remained open. Following the cell fate of hypertrophic chondrocytes by genetic lineage tracing using BACCol10;Cre induced YFP-reporter gene expression we show that a progeny of Col10Cre-reporter labelled osteoprogenitor cells and osteoblasts appears in the primary spongiosa and participates – depending on the developmental stage – substantially in trabecular, endosteal, and cortical bone formation. YFP+ trabecular and endosteal cells isolated by FACS expressed Col1a1, osteocalcin and runx2, thus confirming their osteogenic phenotype. In searching for transitory cells between hypertrophic chondrocytes and trabecular osteoblasts we identified by confocal microscopy a novel, small YFP+Osx+ cell type with mitotic activity in the lower hypertrophic zone at the chondro-osseous junction. When isolated from growth plates by fractional enzymatic digestion, these cells termed CDOP (chondrocyte-derived osteoprogenitor) cells expressed bone typical genes and differentiated into osteoblasts in vitro. We propose the Col10Cre-labeled CDOP cells mark the initiation point of a second pathway giving rise to endochondral osteoblasts, alternative to perichondrium derived osteoprogenitor cells. These findings add to current concepts of chondrocyte-osteocyte lineages and give new insight into the complex cartilage-bone transition process in the growth plate.

ABSTRACT: Scoliosis progression in skeletally immature patients depends on remaining growth. Relationships between vertebral growth plate histomorphometry, growth rates, and mechanical stresses have been reported in several animal studies. Hypertrophic zone heights and chondrocyte heights have been used to assess treatments that aim to modulate growth. The purpose of this study was to determine whether human vertebral physeal hypertrophic zone and cell heights differed between two groups: Severe scoliosis and autopsy controls. Severity was defined at time of surgical planning by curve magnitude and curve stiffness. Physeal samples were obtained from the convex side apex, and from the concave side when feasible. Histologic sections were prepared, and digital images were used to measure hypertrophic zone height, cell height, and cell width. Thirteen spinal deformity patients were included, mean curve magnitude 67° (±23). Etiologies were juvenile and adolescent idiopathic, congenital, neurofibromatosis, neuromuscular, and Marfan syndrome. Five age‐matched autopsy specimens without scoliosis served as controls. Results were presented by etiology, then all convex scoliosis specimens were combined and compared to controls. Zone heights for scoliosis, convex side, and controls were 152 µm (±34) and 180 µm (±42) (p = 0.21), cell heights 8.5 µm (±1.1) and 12.8 µm (±1.2) (p < 0.0005), and cell widths 14.9 µm (±1.5) and 15.0 µm (±2.5), respectively. Human values were compared to published animal models and to a quantitative theory of a stress ̶ growth curve. This quantification of vertebral physeal structures in scoliosis may be expected to help assess theories of progression and potential treatments using growth modulation. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2450–2459, 2018. [ABSTRACT FROM AUTHOR]

Collagen from marine organisms has a broad prospect in biomedical field, yet the knowledge on marine-derived type II collagen is rare. Herein, a novel type II collagen was successfully isolated from squid cartilage for the first time. After being characterized, the immunogenicity of squid type II collagen (SCII) was evaluated and compared with that of bovine type II collagen (BCII). Then investigations were further conducted for the impacts of SCII on pro-inflammatory macrophages and macrophage chemotaxis. The degenerative osteoarthritis (OA) -relieving effects of SCII were explored using OA rat model in vivo . Our results demonstrated that the isolated SCII maintained triple-superhelical structure of native collagen with high purity. Different from BCII, SCII presented no immunogenicity since it neither induced abnormal proliferation of lymphocytes in vitro nor changed the basic levels of IgM, IgG, anti-type II collagen IgG and CD4 + /CD8 + lymphocytes ratio in vivo . Additionally, SCII also exerted prominent anti-inflammatory effects. SCII significantly reduced the production of pro-inflammatory cytokines by enhancing the activity of TCPTP and subsequently prompting the dephosphorylation of p-STAT1 in pro-inflammatory macrophages. Besides, it indirectly prevented hypertrophic changes of chondrocytes, and markedly impeded chemotaxis of macrophages. Moreover, inflammation condition in OA rats was significantly alleviated under treatment with SCII. These data suggested that the newly developed SCII could not only avoid the immunogenic risks of collagen derived from terrestrial animals, but more importantly, provide new choice for the control and treatment of OA. [ABSTRACT FROM AUTHOR]

An autosomal-recessive inactivating mutation R272Q in the human intestinal cell kinase (ICK) gene caused profound multiplex developmental defects in human endocrine-cerebro-osteodysplasia (ECO) syndrome. ECO patients exhibited a wide variety of skeletal abnormalities, yet the underlying mechanisms by which ICK regulates skeletal development remained largely unknown. The goal of this study was to understand the structural and mechanistic basis underlying skeletal anomalies caused by ICK dysfunction. Ick R272Q knock-in transgenic mouse model not only recapitulated major ECO skeletal defects such as short limbs and polydactyly but also revealed a deformed spine with defective intervertebral disk. Loss of ICK function markedly reduced mineralization in the spinal column, ribs, and long bones. Ick mutants showed a significant decrease in the proliferation zone of long bones and the number of type X collagen-expressing hypertrophic chondrocytes in the spinal column and the growth plate of long bones. These results implicate that ICK plays an important role in bone and cartilage development by promoting chondrocyte proliferation and maturation. Our findings provided new mechanistic insights into the skeletal phenotype of human ECO and ECO-like syndromes. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is the most common disease of the joints, and is characterized by the breakdown of cartilage and degradation of the extracellular matrix. OA causes a high level of patient suffering and incurs large societal costs; however, the current strategies for treating OA are restricted due to limited understanding of the underlying molecular and cellular mechanisms. In the present study, the beneficial effects of isoimperatorin (Iso) were investigated using an experimental mouse model of OA, and its mechanism of action on primary chondrocytes was elucidated. Destabilization of the medial meniscus was performed on 8-week-old male mice to induce OA in the knees. Iso (500 mg/g/day) was intragastrically administered for 4 weeks. Degeneration of articular cartilage was assessed by histology using the Osteoarthritis Research Society International scoring system. The expression of matrix metalloproteinase (MMP)13, Runt-related transcription factor (Runx)2, type X collagen (Col X) and vascular endothelial growth factor (VEGF) in the knee joints was examined by immunohistochemistry. In vitro, murine primary chondrocytes were treated with various concentrations of Iso, followed by 10 ng/ml interleukin-1. The mRNA expression levels of MMP13, Runx2, Col X and VEGF were determined by reverse transcription-quantitative polymerase chain reaction. The levels of autophagy and mammalian target of rapamycin (mTOR) signaling were determined by western blotting. Iso significantly ameliorated the severity of articular cartilage degradation in mice with experimental OA. The expression levels of MMP13, Runx2, Col X and VEGF were reduced in Iso-treated mice. In murine primary chondrocytes, Iso also reduced MMP13, Runx2, Col X and VEGF expression, and activated autophagy by downregulating the mTOR complex 1 (mTORC1) signaling pathway. Therefore, the results of the present study demonstrated that Iso ameliorates OA-induced pathological alterations by delaying chondrocyte deterioration, activating autophagy and inhibiting mTORC1, which suggests that Iso may have therapeutic potential for attenuating articular cartilage degradation and treating OA. [ABSTRACT FROM AUTHOR]

Summary The abundant proteoglycan, aggrecan, is resorbed from growth plate cartilage during endochondral bone ossification, yet mice with genetically-ablated aggrecan-degrading activity have no defects in bone formation. To account for this apparent anomaly, we propose that lysosomal hydrolases degrade extracellular, hyaluronan-bound aggrecan aggregates in growth plate cartilage, and that lysosomal hydrolases are released from hypertrophic chondrocytes into growth plate cartilage via Ca2+-dependent lysosomal exocytosis. In this study we confirm that hypertrophic chondrocytes release hydrolases via lysosomal exocytosis in vitro and we show in vivo evidence for lysosomal exocytosis in hypertrophic chondrocytes during skeletal development. We show that lysosome-associated membrane protein 1 (LAMP1) is detected at the cell surface following in vitro treatment of epiphyseal chondrocytes with the calcium ionophore, ionomycin. Furthermore, we show that in addition to the lysosomal exocytosis markers, cathepsin D and β-hexosaminidase, ionomycin induces release of aggrecan- and hyaluronan-degrading activity from cultured epiphyseal chondrocytes. We identify VAMP-8 and VAMP7 as v-SNARE proteins with potential roles in lysosomal exocytosis in hypertrophic chondrocytes, based on their colocalisation with LAMP1 at the cell surface in secondary ossification centers in mouse tibiae. We propose that resorbing growth plate cartilage involves release of destructive hydrolases from hypertrophic chondrocytes, via lysosomal exocytosis.

Sclerostin is a 24-kDa secreted glycoprotein that has been identified as a negative modulator of new bone formation and may play a major role in age-related decline in skeletal function. Although serum levels of sclerostin markedly increase with age, relatively little is known about whether cells in the skeleton change their expression of sclerostin with aging. Using immunohistochemistry and confocal microscopy, we explored sclerostin immunoreactivity (sclerostin-IR) in the femurs of 4-, 9-, and 24-month-old adult C3H/HeJ male mice. In the femur, the only two cell types that expressed detectable levels of sclerostin-IR were bone osteocytes and articular cartilage chondrocytes. At three different sites along the diaphysis of the femur, only a subset of osteocytes expressed sclerostin-IR and the percentage of osteocytes that expressed sclerostin-IR increased from approximately 36% to 48% in 4- vs. 24-month-old mice. In marked contrast, in the same femurs, there were ~40% fewer hypertrophic chondrocytes of articular cartilage that expressed sclerostin-IR when comparing 24- vs. 4-month-old mice. Understanding the mechanism(s) that drive these divergent changes in sclerostin-IR may provide insight into understanding and treating the age-related decline of the skeleton. [ABSTRACT FROM AUTHOR]

The terminal differentiation of hypertrophic chondrocytes is a tightly regulated process that plays a pivotal role in endochondral ossification. As a negative regulator, Sox9 is essentially down regulated in terminally differentiated hypertrophic chondrocytes. However, the underlying mechanismof Sox9 silencing is undefined. Here we show that the zinc finger protein Zbtb20 regulates the terminal differentiation of hypertrophic chondrocytes by repressing Sox9. In the developing skeleton of the mouse, Zbtb20 protein is highly expressed by hypertrophic chondrocytes from late embryonic stages. To determine its physiological role in endochondral ossification, we have generated chondrocyte-specific Zbtb20 knockout mice and demonstrate that disruption of Zbtb20 in chondrocytes results in delayed endochondral ossification and postnatal growth retardation. Zbtb20 deficiency caused a delay in cartilage vascularization and an expansion of the hypertrophic zone owing to reduced expression of Vegfa in the hypertrophic zone. Interestingly, Sox9, a direct suppressor of Vegfa expression, was ectopically up regulated at both mRNA and protein levels in the late Zbtb20-deficient hypertrophic zone. Furthermore, knockdown of Sox9 greatly increased Vegfa expression in Zbtb20-deficient hypertrophic chondrocytes. Our findings point to Zbtb20 as a crucial regulator governing the terminal differentiation of hypertrophic chondrocytes at least partially through repression of Sox9. [ABSTRACT FROM AUTHOR]

Endochondral bone formation is an important pathway in fracture healing, involving the formation of a cartilaginous soft callus and the process of cartilage-to-bone transition. Failure or delay in the cartilage-to-bone transition causes an impaired bony union such as nonunion or delayed union. During the healing process, multiple types of cells including chondrocytes, osteoprogenitors, osteoblasts, and endothelial cells coexist in the callus, and inevitably crosstalk with each other. Hypertrophic chondrocytes located between soft cartilaginous callus and bony hard callus mediate the crosstalk regulating cell-matrix degradation, vascularization, osteoclast recruitment, and osteoblast differentiation in autocrine and paracrine manners. Furthermore, hypertrophic chondrocytes can become osteoprogenitors and osteoblasts, and directly contribute to woven bone formation. In this review, we focus on the roles of hypertrophic chondrocytes in fracture healing and dissect the intermingled crosstalk in fracture callus during the cartilage-to-bone transition., Competing Interests: Joe Kodama: No conflict of interest to report. Kevin J. Wilkinson: No conflict of interest to report. Masahiro Iwamoto: No conflict of interest to report. Satoru Otsuru: Funded by 10.13039/100000002NIH Grant Number R01AR075733, R21AR077654. Motomi Enomoto-Iwamoto: No conflict of interest to report. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors., (© 2022 The Authors.)

Abstract: Transforming growth factor β (Tgfb) signaling plays an important role in endochondral ossification. Previous studies of mice in which the Tgfb type II receptor gene (Tgfbr2) was deleted in the limb bud mesenchymal cells or differentiated chondrocytes showed defects in the development of the long bones or the axial skeleton, respectively. Here, we generated mouse embryos in which the Tgfbr2 gene was ablated in hypertrophic chondrocytes. These mice exhibited delays in both the hypertrophic conversion of proliferating chondrocytes and the subsequent terminal chondrocyte differentiation. The expression domains of Col10a1, Matrix metalloproteinase 13, and Osteopontin were small, and the expression of Vascular endothelial growth factor and Platelet endothelial cell adhesion molecule was downregulated. The calcification of the bone collar in the mutant mice was markedly delayed and the periosteum was thin, possibly because of the downregulation of Indian hedgehog expression. We conclude that Tgfb signaling in hypertrophic chondrocytes positively regulates terminal chondrocyte differentiation, angiogenesis in calcified cartilage, and osteogenesis in the bone collar, at least partly through Indian hedgehog signaling in vivo. [Copyright &y& Elsevier]

Summary: Objective: To identify the molecular differences between the transient and permanent chondrocyte phenotype in osteophytic and articular cartilage. Methods: Total RNA was isolated from the cartilaginous layer of osteophytes and from intact articular cartilage from knee joints of 15 adult human donors and subjected to cDNA microarray analysis. The differential expression of relevant genes between these two cartilaginous tissues was additionally validated by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and by immunohistochemistry. Results: Among 47,000 screened transcripts, 600 transcripts were differentially expressed between osteophytic and articular chondrocytes. Osteophytic chondrocytes were characterized by increased expression of genes involved in the endochondral ossification process [bone gamma-carboxyglutamate protein/osteocalcin (BGLAP), bone morphogenetic protein-8B (BMP8B), collagen type I, alpha 2 (COL1A2), sclerostin (SOST), growth arrest and DNA damage-induced gene 45ß (GADD45ß), runt-related transcription factor 2 (RUNX2)], and genes encoding tissue remodeling enzymes [matrix metallopeptidase (MMP)9, 13, hyaluronan synthase 1 (HAS1)]. Articular chondrocytes expressed increased transcript levels of antagonists and inhibitors of the BMP- and Wnt-signaling pathways [Gremlin-1 (GREM1), frizzled-related protein (FRZB), WNT1 inducible signaling pathway protein-3 (WISP3)], as well as factors that inhibit terminal chondrocyte differentiation and endochondral bone formation [parathyroid hormone-like hormone (PTHLH), sex-determining region Y-box 9 (SOX9), stanniocalcin-2 (STC2), S100 calcium binding protein A1 (S100A1), S100 calcium binding protein B (S100B)]. Immunohistochemistry of tissue sections for GREM1 and BGLAP, the two most prominent differentially expressed genes, confirmed selective detection of GREM1 in articular chondrocytes and that of BGLAP in osteophytic chondrocytes and bone. Conclusions: Osteophytic and articular chondrocytes significantly differ in their gene expression pattern. In articular cartilage, a prominent expression of antagonists inhibiting the BMP- and Wnt-pathway may serve to lock and stabilize the permanent chondrocyte phenotype and thus prevent their terminal differentiation. In contrast, osteophytic chondrocytes express genes with roles in the endochondral ossification process, which may account for their transient phenotype. [ABSTRACT FROM AUTHOR]

Mammalian bones have three distinct origins (paraxial mesoderm, lateral plate mesoderm, and neural crest) and undergo two different modes of formation (intramembranous and endochondral). Bones derived from the paraxial mesoderm and lateral plate mesoderm mainly form through the endochondral process. During this process, hypertrophic chondrocytes play a vital role in inducing osteogenesis. So far, a number of published papers have provided evidence that chondrocyte hypertrophy and osteoblast differentiation are controlled by a variety of signaling pathways and factors; however, little is known about their hierarchy (which are upstream? which are most potent?). In this review, we discuss the signaling pathways and transcriptional factors regulating chondrocyte hypertrophy and osteoblast differentiation based on the evidence that has been reported and confirmed by multiple independent groups. We then discuss which factor would provide the most coherent evidence for its role in endochondral ossification. [ABSTRACT FROM AUTHOR]

Abstract: Hypertrophic chondrocytes exist in two forms detectable by electron microscopy, light and dark chondrocytes; the functional implications of the heterogeneous morphology are unknown. The aims of the study were to establish a method for separating light from dark hypertrophic chondrocytes and to identify genes differentially expressed between the two populations. Three-dimensional pellet cultures of chondrocytes from cartilage of neonatal rats were induced to undergo hypertrophy by treatment with triiodothyronine. Cultures were dissociated and subjected to density gradient centrifugation. The cell fraction with the lowest density comprised predominantly light hypertrophic chondrocytes, and the fraction with the highest density comprised predominantly dark hypertrophic chondrocytes. An Affymetrix GeneChip® rat expression array was used to compare expression between dark cell-containing pellets and the light cell-enriched fraction. Genes identified on the array as putative dark cell-selective genes included genes encoding extracellular matrix proteins and enzymic modulators thereof. Expression of a subset of genes (Col1a1, periostin, osteoglycin, tPA/Plat, and Chst11) was confirmed as dark cell-selective using quantitative reverse transcriptase polymerase chain reaction. The most highly differentially expressed dark cell-selective gene was periostin. In immunocytochemical studies of light and dark cell-enriched fractions, periostin staining was detectable in dark, but not light hypertrophic chondrocytes. The results provide insight into molecular differences between light and dark hypertrophic chondrocytes. [Copyright &y& Elsevier]

Abstract: We have studied hypertrophic and immediately adjacent pre-hypertrophic chondrocytes at the same stage of histologic development in 7day old post-natal Balb/C mouse physes and epiphyses. Laser capture microdissection (LCM) and GeneChip microarray analysis compared the molecular composition of the two hypertrophic chondrocyte regions. Molecules upregulated in dramatically higher levels in the epiphysis were gremlin (58-fold), epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (25-fold), and frizzled related protein (6.4-fold and 5.7-fold). Molecules upregulated in higher levels in the physis were proline arginine-rich end leucine-rich repeat protein (PRELP) (15.6-fold), pyrophosphatase (inorganic) 1 (10-fold) and hedgehog-interacting protein (7.3-fold). Immunocytochemistry for gremlin confirmed specific localization patterns. This study indicates a critical site-specific role for hypertrophic chondrocytes with different synthesis patterns in separate regions even though they appear structurally the same and are at the same stage of development. [Copyright &y& Elsevier]

The role of Notch signaling in cartilage differentiation and maturation in vivo was examined. Conditional Notch pathway gain and loss of function was achieved using a Cre/loxP approach to manipulate Notch signaling in cartilage precursors and chondrocytes of the developing mouse embryo. Conditional overexpression of activated Notch intracellular domain (NICD) in the chondrocyte lineage results in skeletal malformations with decreased cartilage precursor proliferation and inhibited hypertrophic chondrocyte differentiation. Likewise, expression of NICD in cartilage precursors inhibits sclerotome differentiation, resulting in severe axial skeleton abnormalities. Furthermore, conditional loss of Notch signaling via RBP-J gene deletion in the chondrocyte lineage results in increased chondrocyte proliferation and skeletal malformations consistent with the observed increase in hypertrophic chondrocytes. In addition, the Notch pathway inhibits expression of Sox9 and its target genes required for normal chondrogenic cell proliferation and differentiation. Together, our results demonstrate that appropriate Notch pathway signaling is essential for proper chondrocyte progenitor proliferation and for the normal progression of hypertrophic chondrocyte differentiation into bone in the developing appendicular and axial skeletal elements. [ABSTRACT FROM AUTHOR]

The article reports on the results of research which was conducted in an effort to identify and localize the Cis-enhancer element for mouse type X collagen expression in hypertrophic chondrocytes in vivo. Researchers were able to locate a 90-bp ds-enhancer required for tissue- specific Coll10al expression in vivo and putative DNA/protein complexes that contribute lo the regulation of chondrocyte hypertrophy.

Abstract: Chick and mouse embryos with heritable deficiencies of aggrecan exhibit severe dwarfism and premature death, demonstrating the essential involvement of aggrecan in development. The aggrecan-deficient nanomelic (nm) chick mutant E12 fully formed growth plate (GP) is devoid of matrix and exhibits markedly altered cytoarchitecture, proliferative capacity, and degree of cell death. While differentiation of chondroblasts to pre-hypertrophic chondrocytes (IHH expression) is normal up to E6, the extended periosteum expression pattern of PTCH (a downstream effector of IHH) indicates altered propagation of IHH signaling, as well as accelerated down-regulation of FGFR3 expression, decreased BrdU incorporation and higher levels of ERK phosphorylation, all indicating early effects on FGF signaling. By E7 reduced IHH expression and premature expression of COL10A1 foreshadow the acceleration of hypertrophy observed at E12. By E8, exacerbated co-expression of IHH and COL10A1 lead to delayed separation and establishment of the two GPs in each element. By E9, increased numbers of cells express P-SMAD1/5/8, indicating altered BMP signaling. These results indicate that the IHH, FGF and BMP signaling pathways are altered from the very beginning of GP formation in the absence of aggrecan, thereby inducing premature hypertrophic chondrocyte maturation, leading to the nanomelic long bone growth disorder. [Copyright &y& Elsevier]

Abstract: Objective: Previous studies indicate that hypertrophic chondrocytes can transdifferentiate or dedifferentiate and redifferentiate into bone cells during the endochondral bone formation. Mandibular condyle in aged c-src-deficient mice has incremental line-like striations consisting of cartilaginous and non-cartilaginous layers, and the former contains intact hypertrophic chondrocytes in uneroded lacunae. The purpose of this study is to determine the phenotype changes of uneroded hypertrophic chondrocytes. Design: Immunohistochemical and ultrastructural examinations of the pericellular matrix of hypertrophic chondrocytes in the upper, middle, and lower regions of the mandibular condyle were conducted in aged c-src-deficient mice, using several antibodies of cartilage/bone marker proteins. Results: Co-localisation of aggrecan, type I collagen, and dentin matrix protein-1 (DMP-1) or matrix extracellular phosphoprotein (MEPE) was detected in the pericellular matrix of the middle region. Ultrastructurally, granular substances in the pericellular matrix of the middle region were the remains of upper region chondrocytes, which were mixed with thick collagen fibrils. In the lower region, the width of the pericellular matrix and the amount of collagen fibrils were increased. Versican, type I collagen, DMP-1, and MEPE were detected in the osteocyte lacunae. Additionally, DMP-1 and MEPE were detected in the pericellular matrix of uneroded hypertrophic chondrocytes located in the lower, peripheral region of the mandibular condyle in younger c-src-deficient mice, but not in the aged wild-type mice. Conclusions: These results indicate that long-term survived, uneroded hypertrophic chondrocytes, at least in a part, acquire osteocytic characteristics. [Copyright &y& Elsevier]

Abstract: The Maf family of transcription factors is expressed during development of various organs and tissues, and is involved in a variety of developmental and cellular differentiation processes. We previously found that c-maf and mafB are strongly expressed in hypertrophic chondrocytes during cartilage development. Connective tissue growth factor (CTGF) is also expressed in hypertrophic chondrocytes. Adenovirus mediated introduction of c-maf gene into the mouse fibroblast cell line C3H10T1/2 strongly induced CTGF expression. CTGF can be induced by TGF-β via the SMAD pathway; however, the c-Maf could not induce TGF-β, nor could TGF-β induce the c-Maf, suggesting that activation of CTGF by Maf is TGF-β independent. Reporter transfection analysis using C3H10T1/2 cells shows that c-Maf stimulates a CTGF reporter gene. Lc-Maf, a splice variant of c-Maf containing an extra 10 amino acids in the carboxyl terminus, was a stronger inducer of the CTGF reporter gene than c-Maf. Chromatin immunoprecipitation analysis showed that c-Maf binds to the promoter region of the CTGF gene, indicating that Maf directly activates the CTGF gene. Taken together, these data indicate that the CTGF gene is a target of c-Maf and Lc-Maf in cartilage development. [Copyright &y& Elsevier]

Abstract: Runx2 transcribes Runx2-II and Runx2-I isoforms with distinct N-termini. Deletion of both isoforms results in complete arrest of bone development, whereas selective loss of Runx2-II is sufficient to form a grossly intact skeleton with impaired endochondral bone development. To elucidate the role of Runx2-II in osteoblast function in adult mice, we examined heterozygous Runx2-II (Runx2-II+/−) and homozygous Runx2-II (Runx2-II−/−)-deficient mice, which, respectively, lack one or both copies of Runx2-II but intact Runx2-I expression. Compared to wild-type mice, 6-week-old Runx2-II+/− had reduced trabecular bone volume (BV/TV%), cortical thickness (Ct.Th), and bone mineral density (BMD), decreased osteoblastic and osteoclastic markers, lower bone formation rates, impaired osteoblast maturation of BMSCs in vitro, and significant reductions in mechanical properties. Homozygous Runx2-II−/− mice had a more severe reduction in BMD, BV/TV%, and Ct.Th, and greater suppression of osteoblastic and osteoclastic markers than Runx2-II+/− mice. Non-selective Runx2+/− mice, which have an equivalent reduction in Runx2 expression due to the lack one copy of Runx2-I and II, however, had an intermediate reduction in BMD. Thus, selective Runx2-II mutation causes diminished osteoblastic function in an adult mouse leading to low-turnover osteopenia and suggest that Runx2-I and II have distinct functions imparted by their different N-termini. [Copyright &y& Elsevier]

Seven genes preferentially expressed in undifferentiated human bone marrow stromal cells (BMSC) with respect to BMSC-derived osteoblasts were previously identified by differential display. Here we characterize the expression of one of these genes, URB, belonging to the sushi-repeat-containing protein superfamily. In culture, URB is expressed in both human primary and cloned BMSC, and is drastically downregulated during osteoblastic differentiation of these cells. Here we show that in mouse tissues a single 3.8kb Urb transcript is detected and that the mouse Urb protein is secreted as a 150kDa glycoprotein. During mouse development Urb RNA is barely detectable in 9dpc embryos and increases at later stages. Both in situ hybridization and immunohistochemistry analysis show Urb expression in mouse embryos starting from 14dpc mostly in cartilage. The temporal and spatial expression pattern of Urb suggests its role in mouse skeletogenesis. [Copyright &y& Elsevier]

Estrogen plays an important role in the human growth plate by accelerating growth and promoting epiphyseal fusion inbothsexes. Nevertheless, the precise mechanisms responsible for these effects are poorly understood. In the present study, we examined the role of 17ß-estradiol (E2) on cell proliferation and viability, type X collagen synthesis, alkaline phosphatase activity, and matrix calcification in primary cultures of resting, proliferating, and prehypertrophic chondrocytes derived from explants of the bovine fetal epiphyseal growth plate. Growth plate chondrocytes were isolated and separated into maturationally distinct subpopulations, which were cultured for 7-21 days to high density in either (1) serum-free medium, (2) 1 nM thyroid hormone (T3), (3) E2 concentrations ranging from 10-13 M to 10-7 M, or (4) a combination of T3 and E2. To compare E2 effects in both sexes, chondrocytes were harvested from 8 fetuses of both sexes. After hormone treatment, cell cultures were analyzed for cell number and viability, collagen type X, alkaline phosphatase (ALP), and matrix calcification. Neither DNA content nor cell viability were affected by the duration or type of hormone treatment. By itself, E2 stimulated maturation of all subpopulations only in pharmacologic doses (10-7 M). Physiologic E2 concentrations were no different than negative controls treated with ITS (insulin, transferrin, and selenite). Regardless of E2 concentrations, the addition of E2 to 1 nM T3 did not appreciably affect the response to T3 alone, which stimulates maturation of the phenotype. All effects were comparable in both male and female chondrocytes, in all cell subpopulations (maturation stages) and fetuses of varying gestational age. These findings indicate that at physiologic concentrations, the effects of E2 on fetal bovine growth plate chondrocyte appear to be indirect and independent of T3, suggesting that,in vivo, E2 acts in concert with other factors or hormones to induce fusion of the growth plate. [ABSTRACT FROM AUTHOR]

During the initiation of endochondral ossification three events occur that are inextricably linked in time and space: chondrocytes undergo terminal differentiation and cell death, the skeletal vascular endothelium invades the hypertrophic cartilage matrix, and osteoblasts differentiate and begin to deposit a bony matrix. These developmental programs implicate three tissues, the cartilage, the perichondrium, and the vascular endothelium. Due to their intimate associations, the interactions among these three tissues are exceedingly difficult to distinguish and elucidate. We developed an ex vivo system to unlink the processes initiating endochondral ossification and establish more precisely the cellular and molecular contributions of the three tissues involved. In this ex vivo system, the renal capsule of adult mice was used as a host environment to grow skeletal elements. We first used a genetic strategy to follow the fate of cells derived from the perichondrium and from the vasculature. We found that the perichondrium, but not the host vasculature, is the source of both trabecular and cortical osteoblasts. Endothelial cells residing within the perichondrium are the first cells to participate in the invasion of the hypertrophic cartilage matrix, followed by endothelial cells derived from the host environment.We then combined these lineage analyses with a series of tissue manipulations to address how the absence of the perichondrium or the vascular endothelium affected skeletal development. We show that although the perichondrium influences the rate of chondrocytes maturation and hypertrophy, it is not essential for chondrocytes to undergo late hypertrophy. The perichondrium is crucial for the proper invasion of blood vessels into the hypertrophic cartilage and both the perichondrium and the vasculature are essential for endochondral ossification. Collectively, these studies clarify further the contributions of the cartilage, perichondrium, and vascular endothelium to long bone development. [Copyright &y& Elsevier]

Abstract The interstitial collagenases have been suggested to play a critical role in bone formation, remodeling, and cancerogenesis. We have previously shown that during mouse development expression of collagenase-3 (MMP-13) is restricted to bone and cartilage (Gack et al., 1995; Tuckermann et al., 2000) and is affected in mice with altered c-Fos and Cbfa-1 expression (Gack et al., 1994; Porte et al., 1999). In this study, using immunohistochemistry (IHC) and in situ hybridization (ISH) techniques, we have identified cells of the osteoblastic lineage to be the origin of strongly enhanced levels of MMP-13 transcripts in c-fos-induced osteosarcomas. Expression in these cells is further increased in c-fos/c-jun double transgenic mice and paralleled by Cbfa-1 expression. Similarly, in spontaneous and radiation-induced osteosarcomas, both c-Fos and MMP-13 proteins are detectable, suggesting that overexpression of both genes is a characteristic feature of osteosarcomas of different origin. We also observed high levels of MMP-13 in c-Fos-induced chondrosarcomas. In osteoblast-like cells and in cells of late chondrocyte differentiation such as hypertrophic chondrocytes, high levels of MMP-13 transcripts were found. In contrast, in anaplastic areas of the tumors representing highly proliferating chondrocytes, no MMP-13 expression is detectable, suggesting that in addition to Fos/AP-1, bone-specific transcription factors are responsible for restricted expression of collagenase-3/MMP-13 in a specific subset of cells of bone and cartilage in physiology and pathology. [ABSTRACT FROM AUTHOR]

Endochondral ossification in the growth cartilage of long bones from the bullfrog Rana catesbeiana was examined. In stage-46 tadpoles and 1-year-old animals, the hypertrophic cartilage had a smooth contact with the bone marrow and the matrix showed no calcification or endochondral bone formation. In spite of showing no aspects of calcification, the chondrocytes exhibited alkaline phosphatase activity and some of them died by apoptosis. However, matrix calcification and endochondral ossification were observed in 2-year-old bullfrogs. Calcium deposits appeared as isolated or coalesced spherical structures in the extracellular matrix of hypertrophic cartilage. Bone trabeculae were restricted to the central area at the sites where the hypertrophic cartilage surface was exposed to the bone marrow. Cartilage matrix calcification and the formation of bone trabeculae were not dependent on each other. Osteoclasts were involved in calcified matrix resorption. These results demonstrate that the calcification of hypertrophic cartilage and the deposition of bone trabeculae are late events in R. catesbeiana and do not contribute to the development and growth of long bones in adults. These processes may play a role in reinforcing bony structures as the bullfrog gains weight in adulthood. In addition, the deposition of bone trabeculae is not dependent on cartilage matrix calcification. [ABSTRACT FROM AUTHOR]

Bone morphogenetic protein (BMP) partially purified from bovine bone was implanted into the thigh muscles of AKR-strain mice to induce an ectopic endochondral ossification tissue. The extracellular matrix components around the hypertrophic chondrocytes in the induced tissue were examined by electron microscopy and immunohistochemistry for type I collagen. At 14 days after implantation, chondrocytes became hypertrophic with calcifying matrix around them. Some hypertrophic chondrocytes showed no degenerative appearances. They were ringed by a pericellular matrix with no metachromasia which was distinct from the metachromatic cartilage matrix. The localization of type I collagen was demonstrated by immuno-staining in this matrix. Ultrastructurally, ring-shaped matrix consisted of interwoven collagen fibrils on which a D-periodic banding pattern could be discerned. Immunoelectron microscopy demonstrated immunoreactivity of type I collagen on these fibrils. These results suggest that some of the BMP-induced hypertrophic chondrocytes maintain their activity and produce type I collagen-rich matrix before their lacunae are eroded. [ABSTRACT FROM PUBLISHER]

The secondary ossification center of 14- to 16-day-old hamster tibiae was examined immunohistochemically with active and inactive Cls-specific antibodies, RK5 and RK4, respectively. At the ossification center, chondrocytes differentiate from proliferating and hypertrophic to degenerating stages, and their site is occupied by the bone marrow. Cls was strongly immunostained in hypertrophic chondrocytes. In order to discover whether Cls is activated at a particular site, the cartilage was immunostained with RK5 and RK4. RK5 mainly reacted with degrading matrix around invading vessels. In contrast, RK4 strongly stained hypertrophic chondrocytes. Immunoelectron microscopy revealed Cls on degrading fragments of chondrocytes and fibers of cartilage matrix. Decorin, one of the major matrix proteoglycans, was dose and time dependently degraded by Cls. Type II collagen and type I gelatin were also degraded. Articular cartilage from patients with rheumatoid arthritis was positively immunostained (11/12 cases) with an anti-Cls monoclonal antibody (mAb) PG11, whereas normal articular cartilage (5/5 cases) was negative, suggesting Cls participation in the etiology of rheumatoid arthritis. [ABSTRACT FROM AUTHOR]

Skeletal development is a highly sophisticated process in which the expression of a variety of growth factors, signaling molecules, and extracellular matrix proteins is spatially and temporally orchestrated. In the present study, we show that ADAM10, a transmembrane protease that is critically involved in the functional regulation of various membrane-bound molecules, plays an essential role in the longitudinal growth of long bones and in skeletal development. We found that mutant mice lacking ADAM10 in osteochondroprogenitors exhibited marked growth retardation and had shorter long bones than the control mice. Histomorphometric analysis revealed that the mutant mice had a shorter hypertrophic zone and that their hypertrophic chondrocytes were smaller in size than those of the control mice. Unexpectedly, we found that the mRNA expression of the chemokine CXCL12 and its receptor CXCR4 were significantly reduced in cartilage tissues lacking ADAM10. Further, exogenous supplementation of recombinant CXCL12 rescued the defect in the ADAM10-deficient growth plate in an ex vivo culture model. Taken together, our data show a previously unknown role for ADAM10 in skeletal development that involves its regulation of the CXCL12 and CXCR4 signaling pathway. • Conditional abrogation of ADAM10 in osteochondroprogenitors in mice (Adam10 ΔCol2 mice) leads to dwarfism. • Longitudinal growth rate of long bones is decreased in Adam10 ΔCol2 mice. • Growth plates of Adam10 ΔCol2 mice are shorter and the size of hypertrophic chondrocytes is smaller than the control animals • The expression of CXCL12 and CXCR4 transcripts is reduced in Adam10 ΔCol2 mice cartilage tissues. • Exogenous supplementation of CXCL12 rescues the defects in Adam10 ΔCol2 growth plates in an ex vivo organ culture model. [ABSTRACT FROM AUTHOR]